The membrane-bound NADPH oxidase in phagocytes, gp91(phox) (a.k.a. Nox2), produces superoxide, a precursor of microbicidal oxidants, thereby playing a crucial role in host defense. Activation of gp91(phox)/Nox2 requires assembly with the cytosolic proteins p67(phox) and p47(phox), each containing two SH3 domains. Although the C-terminal SH3 domain of p67(phox) is responsible for binding to p47(phox), little is known about the role for the first (N-terminal) SH3 domain [SH3(N)]. Here we show that truncation of p67(phox)-SH3(N), but not substitution of arginine for the invariant residue Trp-277 in SH3(N), results in an impaired activation of gp91(phox)/Nox2. The impairment is overcome by higher expression of an SH3(N)-defective p67(phox) in cells, suggesting that SH3(N) primarily increases the affinity of p67(phox) for the oxidase complex. On the other hand, p67(phox)-SH3(N) is not involved in activation of Nox1 and Nox3, closely-related homologues of gp91(phox)/Nox2. Thus p67(phox)-SH3(N) specifically functions in gp91(phox)/Nox2 activation probably via facilitating oxidase assembly.